Methods for producing parthenocarpic or female sterile transgenic plants and methods for enhancing fruit setting and development

ABSTRACT

Described is the use of the promoter region of the DefH9 gene of  Anthirrhinum majus  or of a promoter of a homologous gene displaying the same expression pattern and characteristics for the establishment of parthenocarpy or female sterility in plants, for increasing gynogenesis or for enhancing fruit setting and development. Also described are recombinant DNA molecules comprising a DefH9 promoter in combination with a DNA sequence which upon expression in plants leads to the above mentioned effects. Furthermore, described are plant cells and plants transformed with such recombinant DNA molecules.

This application is a 371 of PCT/EP97/07202 filed Dec. 19, 1997.

FIELD OF THE INVENTION

The present invention relates to the use of the promoter region of theDefH9 gene of Antirrhinum majus and of promoter regions of homologousgenes in other species for the highly specific expression of genes inthe placenta and/or ovules of plants or tissues derived from placenta orovules in order to achieve, for example, parthenocarpy, female sterilityand for an enhancement in fruit setting and development. The presentinvention also relates to DNA constructs in which said promoter controlsthe expression of a DNA sequence which upon expression leads to theabove mentioned effects. Furthermore, the present invention relates totransgenic plants genetically modified with such constructs which candevelop fruits also in the absence of fertilization (i.e. withparthenocarpic development) or which are female sterile as well as tothe fruits of these plants and to the propagation material of theseplants.

In the field of crop plants grown for the commercial value of theirfruits, there is a great demand for plants able to develop fruits in theabsence of fertilization. This is due not only to the absence of seeds(e.g. table grape, melon), but most prominently, to obtain fruits inenvironmental conditions not favorable for fertilization (e.g. eggplant,tomato; Lipari and Paratore, Acta Hort. 229 (1988), 307-312; Savin, PHMRevue Horticole 374 (1996), 50-52). Methods to achieve parthenocarpicdevelopment essentially consist either in using chemically activeingredients, in using mutants conferring parthenocarpic development tothe species where they have been selected or by using alterations inchromosome number (i.e. polyploidy). Thus, plants which are suitable forbreeding plants with the mentioned desired properties have so far beenavailable only to a limited degree. Indeed, it is common practice forsome horticultural plants to treat (i.e. spray) flower buds withsynthetic growth factors to cause parthenocarpic development (referencesabove, and: La Torre and Imbroglini, Informatore Agrario 16 (1992),71-78; Roberts and Hooley, Plant Growth Regulators, Chapman and Hall(New York (1988)). The success of exogenous application of phytohormonesrelies on their even action on the plant organ, it is labor-intensiveand adds extra costs to the production process. Secondly, the chemicalscan be transported from the site of application, and so they can affectother parts of the plant or the whole plant.

Plant genetic engineering has recently been applied in order tocircumvent the above-mentioned drawbacks connected with the use ofmutants or the exogenous application of phytohormones to the plants. Forexample, Barg, Acts of the III I.S.H.S. Symposium on “In vitro cultureand horticultural breeding” Jerusalem, Jun. 16-21 (1996), 13, disclosedthe generation of transgenic tomato plants which contain a rolB geneunder the control of the TPRP-F1 promoter (from tomato). The use of thispromoter leads to the expression of the rolB gene preferentially in theovary and young fruit. As a result the plants showed parthenocarpicdevelopment. However the promoter used displays also a well detectablelevel of expression in vegetative tissue (i.e. 1.8%, 3.5%, 0.1% in root,stem and leaf, respectively) as compared to expression in the ovary(=100%; Salts et al. (Plant Mol. Biol. 17 (1991), 149-150). Due to thisbasal level of expression, the plant can also be altered in itsphysiological processes in vegetative tissues. In a second example, thesame TPRP-F1 promoter was used by Szechtman et al., Acts of the IIII.S.H.S. Symposium on “In vitro culture and horticultural breeding”Jerusalem, Jun. 16-21 (1996) 32, to drive the expression of thebacterial iaaH gene coding for an indoleacetamide hydrolase able tohydrolyse a number of indoleacetamide analogs, and thus to convert theinactive indolacetamide (IAM) and naphthalenacetamide (NAM) to theactive phytohormones indoleacetic acid (IAA) and napthalene acetic acid(NAA), respectively. The resulting transgenic plants showedparthenocarpic development when sprayed with NAM. This disclosurerepresents an improvement of the efficiency of parthenocarpicdevelopment, but still depends on the exogenous application of chemicalssuch as NAM. The transgene caused no adverse pleiotropic effects per se,though young plants sprayed with 25 ppm NAM have been reported toexhibit a slight epinastic response (Szechtman et al., loc. cit.). Inthe same communication Szechtman et al. (1996) proposed that theTPRP-F1-iaaH system will have to be combined with TPRP-F1-iaaM to enableendogenous auxin biosynthesis in the fruit.

However, the TPRP-F1 promoter used in the disclosed chimeric genes, hasthe drawback that due to its basal level of expression also invegetative tissue it is also active after transformation and during theregeneration process of transformed cells. Since the expression of theiaaM gene or of genes leading to a higher sensitivity for auxins (likerolB) interferes with the regeneration process, the TPRP-F1 promoter isnot suitable to obtain optimal plants transgenic for the iaaM or rolBgene. The reason for this is its constitutive basal level of expressionin vegetative tissue. This feature of the promoter hinders the efficientregeneration of plants with an optimum level of expression and unalteredin their vegetative growth. As a consequence either transgenic plantsare regenerated which do not express the iaaM gene and/or plants areregenerated with a level of constitutive expression of the iaaM or rolBgene so low to be compatible with regeneration. It is known that theconstitutive expression of the iaaM gene has deleterious effects intransgenic plants (Gaudin et al., Plant Physiol. Biochem. 32 (1994),11-29). Thus, these plants do not represent optimal products since i)they might be altered in their vegetative growth (auxin affects manyphysiological processes including interactions with environmental andmicrobial factors) and ii) their level of expression in the ovary mightbe curtailed and not strong enough to promote parthenocarpic developmentefficiently. In the case of plants obtained using transformation methodsnot involving manipulations in tissue culture, the constitutive level ofexpression in vegetative tissues would furthermore interfere with seedgermination and seedling growth. The above described experiments for thegeneration of transgenic plants are based on the well known fact thatdeveloping ovules are a good source of auxins (Archbold and Dennis, J.Amer. Soc. Hort. Sci. 110 (1985), 816-820), and that exogenous auxin cansubstitute the developing ovules (e.g. the achenes of strawberries) tosupport growth of the receptacle (Nitsch, Amer. J. Bot. 37 (1950),211-215; Archbold and Dennis, 1985, loc. cit.) thereby leading toparthenocarpic development. Thus, to mimic the hormonal effects ofpollination by plant genetic engineering, the expression of a chimericgene able to alter auxin content and activity should take placespecifically in cells of the female reproductive organs, preferably inthe ovules and most preferably also in tissue derived therefrom. Thisrequires a promoter which is highly specific for expression in suchcells.

With regard to female sterility there are several reports in the art howto obtain female sterile plants by genetically engineering plants withgenetic information which leads to the killing or disabling of cells ofthe female reproductive organs. These approaches are mainly based on theconcept that a highly toxic agent is produced in the cells, such as anRNase, a protease or a bacterial toxin. Alternatively, antisense RNA ora ribozyme against transcripts of essential genes are produced. Allthese approaches require the highly specific expression of theintroduced construct in cells of the female reproductive organ and theabsence of expression in other tissues since the expression in othertissues would be deleterious for the development of the plant.

Thus, the technical problem underlying the present invention is theprovision of methods and means for the production of transgenic plantswhich due to the highly specific expression of a transgene in cells ofthe female reproductive organ, in particular in the placenta and/or theovule, are female sterile or show a parthenocarpic development or anenhanced parthenocarpic development.

This technical problem is solved by the provision of the embodimentscharacterized in the claims.

SUMMARY OF THE INVENTION

Thus, the present invention relates to the use of a DefH9 promoter forthe expression of a DNA sequence in plant cells for a purpose whichrequires highly specific expression in the placenta and/or ovule of atransgenic plant and an extremely low level of expression in cells ofvegetative tissue. Examples for such purposes are the establishment ofparthenocarpy or female sterility by expressing specific DNA sequencesin cells of the female reproductive organ of transgenic plants. Furtherpurposes are the increase in the rate of gynogenesis and the productionof haploid-double haploid lines or hybridization between distant plantspecies.

Accordingly, in a first aspect the present invention relates to the useof the promoter region of the DefH9 gene of Antirrhinum majus or of apromoter region of a gene homologous to the DefH9 gene of Antirrhinummajus and capable of directing placenta and/or ovule specific expressionof a DNA sequence linked to it in plants for the establishment ofparthenocarpy.

The invention is based on the finding that the promoter region drivingexpression of the DefH9 gene of Antirrhinum majus has an extremely lowlevel of basal activity in tissues other than the placenta and/or theovule. Thus, this promoter is on the one hand suitable to obtain highlyspecific expression of a DNA sequence in the above-mentioned tissues offemale reproductive organs of plants. This helps to avoid unwantedmodifications of other physiological and developmental processes whichconstitute the drawbacks of the methods known in the art. In order toachieve parthenocarpic development, the basal level of constitutiveexpression of the introduced chimeric gene which affects, for example,the auxin metabolism has to be as low as possible (i.e. preferentiallybelow detection limit). This aspect is particularly relevant when thegenetic information introduced into the plant cells controls the leveland activity of growth factors, such as the phytohormones belonging tothe auxin type as is the case when trying to establish parthenocarpy byincreasing the auxin content or activity.

On the other hand this promoter, due to its extremely low level of basalactivity in vegetative tissue helps to circumvent the problems describedabove which arise when using, for example, the TPRP-F1 promoter. Inparticular, since the DefH9 promoter is not active during theregeneration of transformed plant cells to transgenic plants, there isno selection for cells which have only a very low level of promoteractivity. Thus, this promoter is particularly suited and useful for theuse in the generation of transgenic plants which show a parthenocarpicdevelopment due to the expression of a foreign DNA sequence specificallyin cells of the female reproductive organ, namely in the placenta and/orovule and most preferably in tissue derived from placenta or ovule, suchas the integument of seeds.

In the present invention the term “DefH9 promoter” means the promoterregion of the DefH9 gene of Antirrhinum majus including regulatorysequences which is capable to specifically direct expression in theplacenta and/or the ovules of transgenic plants or in tissue derivedtherefrom and which does not show a detectable level of basal expressionin vegetative tissues. Preferably, the promoter starts to directexpression in these tissues before the onset of anthesis. “Not showing adetectable level of expression” in this context means that expression ofa gene linked to a DefH9 promoter cannot be detected in a Northern blotanalysis in probes of vegetative tissue. Such a Northern blot analysiscan be carried out according to methods known in the art and ispreferably carried out as described in the Examples (see below).

The expression of the DefH9 gene occurs very specifically in theplacenta and in the ovules during early phases of flower development asshown by in situ hybridization. It is shown in the present inventionthat the level of expression of the DefH9 promoter in vegetative tissueis below the detection limit of Northern blot analysis (see FIG. 3). TheDefH9 (Deficiens homologue 9) gene of Antirrhinum majus is described inthe Ph. D. thesis of Rolf Hansen (1993, University of Cologne, Facultyof Mathematics and Sciences). This thesis discloses the cDNA sequence aswell as the genomic sequence of the DefH9 gene including its promoterregion and its regulatory sequences. The DefH9 gene is a MADS box gene,expression of which occurs in early stages specifically in thedeveloping placenta and ovules of the female reproductive organ, andbecomes restricted at later stages to the ovules and the vascularbundles of the placenta. The nucleotide sequence of the DefH9 promoterand regulatory regions is depicted in FIG. 2 and also in SEQ ID No. 1.In the sequence the transcription initiation site is located at position2265. The first intron is located in the transcribed but untranslatedleader and corresponds to nucleotides 2418 to 3462 of the sequence givenin SEQ ID No. 1.

The term “promoter” refers to the nucleotide sequences necessary fortranscription initiation, i.e. RNA polymerase binding, and alsoincludes, for example the TATA box.

The term “regulatory regions” refers to sequences which furtherinfluence the level of expression, for example, in the sense that theyconfer tissue specificity. Such regions can be located upstream of thetranscription initiation site, but can also be located downstream of it,e.g. in transcribed but nontranslated leader sequences, especially inintrons.

In the present invention the term “DefH9 promoter” is used as comprisinga promoter and the regulatory regions necessary to obtain the abovedescribed specificity, namely specific expression in the placenta and/orovule of plants and no detectable expression in the cells of vegetativetissue.

In a preferred embodiment of the present invention the promotercomprises the nucleotides 1 to 2264 of the nucleotide sequence as setforth in SEQ ID No. 1 or a fragment thereof which still confers specificexpression in the placenta and/or ovules of transgenic plants. Mostpreferably, such a fragment comprises besides a promoter furthermorenucleotides 2265 to 3480, which corresponds to the transcribed butuntranslated leader sequence of the DefH9 gene of A. majus and mostpreferably comprises the first intron (nucleotides 2418 to 3462). Theterm “DefH9 promoter” also includes promoter regions and regulatoryregions of a gene from the same or another plant species which ishomologous to the DefH9 gene of A. majus and the promoter region ofwhich has the same tissue specificity as the DefH9 promoter. Suchpromoters are characterized by an extremely low level of activity inorgans and tissues other than cells of the female reproductive organs,namely of cells of the placenta and/or the ovule or of tissue derivedfrom placenta or ovule. Genes homologous to the DefH9 gene of A. majusare genes which encode a protein which is structurally or functionallyhomologous to the DefH9 gene product of A. majus. Structural homologypreferably means that the coding region of the gene shows a sequenceidentity to the coding region of the DefH9 gene of A. majus of at least40%, preferably of at least 60% and more preferably of at least 80%.More preferably, the gene homologous to the DefH9 gene of A. majusencodes a protein belonging to the family of the MADS box proteins andeven more preferred a protein belonging to the AGAMOUS subfamily of theMADS box proteins. Thus, according to the invention promoters from otherspecies can be used that are structurally or functionally homologous tothe DefH9 promoter from Antirrhinum majus, or promoters that display anidentical pattern of expression, in the sense of being not onlyexpressed in the ovules and/or placenta but, and most important, notshowing a detectable level of expression in vegetative tissue. It ispossible for the person skilled in the art to isolate with the help ofthe known DefH9 gene from A. majus corresponding genes from other plantvarieties or plant species. This can be done by conventional techniquesknown in the art, for example, by using the DefH9 gene as ahybridization probe or by designing appropriate PCR primers. It is thenpossible to isolate the corresponding promoter region by conventionaltechniques and test it for its expression pattern. For this purpose itis, for instance, possible to fuse the promoter to a reporter gene, suchas luciferase, and assess the expression of the reporter gene intransgenic plants. The present invention also relates to the use ofpromoter regions which are substantially identical to the DefH9 promoterof Antirrhinum majus or to a promoter of a homologous gene or to partsthereof and which are able to confer specific expression in the placentaand/or ovules in plants without detectable expression in cells ofvegetative tissue.

Such promoters differ at one or more positions from the above-mentionedpromoters but still have the same specificity, namely they comprise thesame or similar sequence motifs responsible for the above-describedtissue specificity. Preferably such promoters hybridize to one of theabove-mentioned promoters, most preferably under stringent conditions.Particularly preferred are promoters which share at least 85%, morepreferably 90% and most preferably 95% sequence identity with one of theabove-mentioned promoters and has the same specificity.

As described above, the DefH9 promoter or a promoter of a homologousgene which displays the same or a similar expression pattern with a verylow basal level of expression in vegetative tissue are particularlyuseful for the generation of plants showing a parthenocarpic developmentby genetic engineering. One approach to achieve parthenocarpicdevelopment in plants is the increase in auxin content and/or activityin cells of the female reproductive organ of a plant, preferably in theplacenta and/or ovule. In general, this can be achieved in differentways. In one embodiment of the present invention an increase in thecontent and/or activity of at least one auxin in the cells of the femalereproductive organ is achieved by expressing in the plants a DNAsequence under the control of the DefH9 promoter, wherein said DNAsequence upon expression leads to an increase in the intracellularcontent or activity of at least one auxin. One meaning of the term“increase in the auxin content and/or activity” is that the synthesisrate of an auxin is increased. This might be achieved, for example, byincreasing the conversion of a metabolite directly into an auxin oralternatively by increasing the synthesis and consequently theconcentration of a precursor of an auxin which is then converted intothe respective auxin. Another meaning of this term is that the releaseof conjugated auxins is enhanced or the conjugation of auxins isprevented or reduced.

The term “auxin” comprises in this context naturally occurring andsynthetic organic substances acting as a phytohormone in the sense thatthey promote elongation of shoots and inhibit elongation of roots,preferably in very low concentrations, most preferably already inconcentrations lower than 10⁻⁶ M. Preferably, an auxin shows at leastone of the following effects on plant development: stimulation of celldivision, of cell elongation, and/or of cell expansion, of apicaldominance, stimulation of xylem differentiation, stimulation of the cellelongation and cell division activity of the cells of the cambium,stimulation of lateral and adventitious root formation, stimulation ofnodulation, of germination, of leaf epinasty, of ovary cell growth, ofparthenocarpy, of the formation of female flowers and of leaf expansion.More particularly, the term “auxin” refers to indole acetic acid (IAA)which is most likely synthesized in plant cells from tryptophane viaindole-3-pyruvate and indole-3-acetaldehyde, and which is degraded viaenzymatically catalyzed oxidation.

However, the term also comprises other naturally occurring compoundswhich act as an auxin and which are derived from indole or from anothercompound, for example, the naturally occurring phenyl acetic acid whichis a non-indolic auxin or 4-(indole-3-yl) butyric acid. Furthermore,this term comprises compounds from organisms other than plants orchemically synthesized compounds which have at least one of the effectson plant development as listed above. An example for such a compound is(2,4-dichlorophenoxy)-acetic acid (2,4-D).

In a preferred embodiment the DNA sequence linked to a DefH9 promotercodes for a polypeptide which is naturally involved in the biosynthesisof at least one auxin in plant cells. The expression of the DNA sequencein plant cells then leads to an increase in the biological (for example,enzymatic) activity of this polypeptide and consequently to the increaseof the content and/or activity of at least one auxin in the cells. Thus,in principle, by this embodiment it is contemplated that the auxincontent and/or activity can be increased in plant cells by increasingthe biosynthesis of at least one auxin due to a stimulation/accelerationof a biosynthetic pathway which naturally occurs in plant cells.

In another preferred embodiment the DNA sequence linked to a DefH9promoter codes for a protein which is naturally not expressed in plantcells and which upon expression in plant cells leads to the synthesis ofat least one auxin or a precursor of an auxin from a metabolite presentin plant cells. Genetic information which may be used in this regard is,for example, present in the genomes of several bacteria. For instance,in many cases of bacteria-induced phytopathogenesis, alterations ofhormone biosynthesis, content and activity play an important role in theinteraction between plants and bacteria. Thus, in a more preferredembodiment of the present invention the DNA sequence linked to the DefH9promoter codes for a bacterial protein, namely a protein which leads inplant cells to the increase of the content or activity of at least oneauxin. Preferably said DNA sequence codes for a protein of a bacteriumfrom the genus Pseudomonas or Agrobacterium. More preferably, said DNAsequence codes for a protein of Pseudomonas syringae or of Agrobacteriumrhizogenes or tumefaciens. One example of a gene which is preferablyused for the purpose of the present invention is the iaaM gene ofPseudomonas syringae pv. savastanoi, the etiological agent of planttumors in olive or oleander trees (Spena et al., Curr. Opinion inBiotechnology 3 (1992), 159-163; Gaudin et al., 1994; loc. cit.). Theneoplastic development is caused by phytohormones synthesized by thebacteria, which are then secreted into the surrounding tissues and causeuncontrolled growth of plant cells. Among the genes involved in thepathogenesis of this type of tumour, the iaaM gene codes for theindoleacetamide monooxigenase, and it is responsible for converting byoxidation the amino acid tryptophan to indoleacetamide. Indoleacetamidehas no particular auxin activity, but it is slowly converted, eitherchemically or enzymatically, by plant hydrolases to IAA, the major formof auxin in plants. Expression of the iaaM gene in transgenic plants isable by itself to cause modification of hormone metabolism and activity,and consequently to modify plant biochemical and developmental processes(Sitbon et al., Plant Physiol. 95 (1991), 480-485). Thus, in order toavoid constitutive expression which might interfere with theregeneration of transgenic plants from tissue culture and to avoid thepossibility that transgenic plants are either altered in theirvegetative growth and/or express only a low level of expressioncompatible with the regeneration process, the iaaM coding sequence is,according to the present invention, placed under the control of a DefH9promoter.

The present invention demonstrates that the iaaM gene of Pseudomonassyringae is able to cause parthenocarpic development in plants whendriven by the DefH9 promoter. It is furthermore shown that thetemporally and spatially precise and specific control of expression ofthe iaaM gene plays a crucial role in the genesis of parthenocarpicdevelopment. Expression under the highly specific and tightly regulatedDefH9 promoter and controlling sequences allows the regeneration oftransgenic plants unmodified in their vegetative growth, but modified infruit setting and development. On the contrary, a basal level ofexpression of the iaaM in vegetative tissues is not compatible withregeneration of transgenic plants unmodified in their vegetative growthand still expressing the iaaM gene with optimum strength in the desiredtissue or organ. The iaaM gene from Pseudomonas syringae subsp.savastanoi is known and its sequence has been published (Yamada et al.,Proc. Natl. Acad. Sci. USA 82 (1985), 6522-6526). According to theinvention genes homologous in function to the iaaM gene of P. syringaemight be used for the purpose of this invention. Such genes which arepreferably also homologous with respect to the nucleotide sequence canbe isolated by the person skilled in the art using known methods, e.g.,the screening of cDNA or genomic libraries with probes designed on thebasis of the iaaM gene of P. syringae and subsequently testing the geneproduct for its biological activity. Such genes with an activity similarto that of the iaaM gene product of P. syringae have been cloned, forinstance, from some strains of Agrobacteria (i.e. A. tumefaciens andrhizogenes; see, for instance, Klee et al., Gene Dev. 1 (1987), 86-96;White et al., J. Bacteriol. 164 (1985), 33-44; Cardarelli et al., Mol.Gen. Genet. 208 (1987), 457-463).

The experimental data provided by the present application furthermoreshow that the expression of the iaaM gene under the control of a DefH9promoter has the unexpected effect that these plants show fruit settingand fruit development even under conditions which represent very adverseclimatic conditions for eggplant fertilization and fruit development.Thus, the present invention provides evidence that the expression of DNAsequence leading to an increase in auxin concentration and/or activityspecifically in cells of the female reproductive organs of plants canlead to fruit setting even under adverse climate conditions. This meansthat in this way an enhancement of fruit setting and fruit developmentis achieved. By this it is meant that in comparison to non-transformedplants the transgenic plants of the present invention are able to setfruit (with or without pollination) and to develop them under climaticconditions which in non-transgenic plants would not allow fertilizationand/or fruit development.

In another preferred embodiment of the present invention the DNAsequence the expression of which is driven by the DefH9 promoter is therolB gene from the A4 Ri plasmid of Agrobacterium rhizogenes, theetiological agent of the hairy-root disease (Spena et al., 1992; loc.cit.; Gaudin et al., 1994; loc. cit.) or a gene from another sourcewhich is functionally equivalent to the rolB gene. The rolB gene is ableby itself to alter plant growth. It codes for a protein possessingβ-glucosidase activity which might hydrolyse the inactiveindolethanol-β-glucoside (IEG) releasing the active auxin indolethanolfrom the inactive glucoside. This undisclosed hypothesis predicts thatthis gene does not directly participate in the synthesis oftransportable growth factors, but that it releases indolethanol from theinactive glucoside. Indolethanol might act by itself or it might beconverted to IAA, the major form of auxin in plants. The sequence of therolB gene from Ri plasmid A4 of A. rhizogenes has been published bySlightom et al. (J. Biol. Chem. 261 (1986), 108-121) and a detaileddescription has been given by Spena et al. (EMBO J. 6 (1987),3891-3899). Genes with similar activity have been cloned from some otherstrains of Agrobacterium rhizogenes, and related sequences could becloned from the genomes of some plant species (e.g. Nicotiana glauca).The present invention provides evidence that the rolB gene is not able,in all cases, to trigger parthenocarpic development by itself, at leastnot in tobacco. Thus, the rolB gene under the control of a DefH9promoter is preferably used in plants which show on their own aparthenocarpic development under specific circumstances, for example,under specific environmental conditions. In these cases the occurrenceof parthenocarpic development can be increased by expressing the rolBgene under the control of a DefH9 promoter.

In a preferred embodiment of the present invention, the rolB gene andthe iaaM gene are used in combination to achieve parthenocarpicdevelopment. This might, for example, either be done by introducing thetwo genes simultaneously or by subsequently transforming plants withthese genes. Alternatively, two plants, one of which comprises the iaaMgene under the control of a DefH9 promoter and the other which comprisesthe rolB gene under the control of a DefH9 promoter may be crossed. Inthese cases it is possible that, even if expression of the DefH9-rolBgene itself is not able to cause parthenocarpic development in allplants of interest, it can increase parthenocarpic development,preferably in conjunction with the DefH9-iaaM chimeric gene. Thisembodiment is preferred, inter alia, for the following reason.Experimental results indicated that the rolB gene product is aβ-glucosidase which might hydrolyze the inactiveindolethanol-β-glucoside and therefore releasing indolethanol, a naturalauxin. An increase of IAA is followed by an increased conversion of IAAto indolethanol, which is in turn converted to indolethanol-β-glucoside.Thus, plants transgenic, for instance, for the DefH9-iaaM gene wouldhave an increase not only in IAA but also in the content of the inactiveindolethanol-β-glucoside providing the substrate for the rolB geneproduct within the ovary. Thus, the expression of the rolB gene in thesame tissues will hydrolyse indolethanol-β-glucoside to the active auxinindolethanol (which in turn can be converted back to IAA; Cohen andBialek, in “The Biosynthesis and Metabolism in Higher Plants, Eds.Crozier and Hillmann, Society for Experimental Biology Seminar 23(1984), 165-181). Thus, the use of the DefH9-rolB construct willreinforce the auxin effect of the DefH9-iaaM gene by increasingprimarily the indolethanol content. A practical consequence of this isthe possibility to screen DefH9-rolB transgenic plants to be used incrosses for their capacity to develop parthenocarpic fruits by chemicaltreatment of the flower buds with indolethanol. The exogenously suppliedindolethanol will have a transient action due to its conversion to theinactive indolethanol-β-glucoside in normal and not expressingtransgenic plants, however the expression of a sufficiently high levelof the rolB protein will hydrolyse the inactive glucoside to the activeaglycone. Consequently the two genes would act in two different ways toincrease auxin content and activity: (1) the DefH9-iaaM chimeric genewill increase the content primarily of IAA via indoleacetamide, (2) theDefH9-rolB will have an auxin effect by releasing primarily indolethanolfrom its glucosides. Thus, its effect will be limited only to plantshaving a high content of indolethanol-β-glucoside. A higher content inindolethanol-β-glucoside can be achieved either by chemically adding IAAor indolethanol, or in transgenic plants by expressing the iaaM gene.

In a further preferred embodiment of the present invention the DNAsequence expression of which is driven by a DefH9 promoter is one codingfor indolepyruvate decarboxylase. Indolepyruvate decarboxylase is thekey enzyme in the indole pyruvic pathway of IAA synthesis in which IAAis synthesized from tryptophane. The conversion of indolepyruvic acid isin this pathway the rate limiting step. A DNA sequence encoding asuitable indolepyruvate decarboxylase has been cloned, for example, fromEnterobacter cloacae (see, e.g., Koga et al., Mol. Gen. Genet. 226(1991), 10-16).

The most widespread auxin present in plants, IAA, is mainly conjugatedthrough its carboxyl group to a variety of amino acids, peptides andcarbohydrates. The conjugates represent approximately 95% of the IAApresent in the cells. It is assumed that these conjugates allow rapidalteration of free IAA concentration. Thus, another approach to increasethe auxin content and/or activity in cells of the female reproductiveorgans of plants would be to express under the control of a DefH9promoter a DNA sequence which upon expression leads to an increasedrelease of an auxin, for example, of IAA, from its conjugated form(s) orto a decrease of the conversion of free auxin into a conjugated form. Anexample for such a DNA sequence is a DNA sequence which hydrolyses theconjugates between IAA and an amino acid, a peptide or a carbohydrate.

In a preferred embodiment the DNA sequence, the expression of which isdriven by a DefH9 promoter, is the ILR1 gene of plants. The proteinencoded by an ILR1 gene has aminohydrolase activity and releases IAAfrom conjugates with amino acids. ILR1 genes are known from differentplants, for example, from Arabidopsis thaliana (Bartel and Fink, Science263 (1995), 1745-1748; GenBank accession no. U23794). DNA sequencesencoding proteins with a similar enzymatic activity may be also obtainedfrom bacteria. Further examples are ILL1 and ILL2 genes coding for ILL1and ILL2 proteins, respectively. Such DNA sequences have been isolated,for example, from Arabidopsis thaliana and are available under GenBankaccession numbers U23795 and U23796, respectively.

As mentioned above, another possibility to increase auxin content and/oractivity in plant cells is to reduce the rate of conversion of free IAAinto its conjugates. This may be achieved by inhibiting expression ofgenes coding for proteins catalyzing this conversion. Possibleapproaches are, for example, the expression of an antisense RNA, aribozyme or of a specific inhibitor. A protein catalyzing such areaction is, for example, the protein encoded by the iaglu gene ofmaize. The corresponding DNA sequence has been cloned (see, forinstance, Szersen et al., Science 265 (1994), 1699). Also possible isthe reduction of the degradation rate of an auxin by inhibiting theexpression of the enzymes which are involved in the degradation of thisauxin, for example, of IAA. A further possibility to increase auxincontent and/or activity is the inhibition of its transport.

Another meaning of the term “increase in the auxin content and/oractivity” is that the activity or sensitivity of an auxin receptor isincreased. Thus, it is, for example, possible to express in plant cellsunder the control of the above-described promoters a protein which is areceptor of auxin and thereby increase the biological activity of auxin.It is also possible to express auxin receptors with a higher sensitivityfor auxin. This would also lead to an increase in the biologicalactivity of auxin.

Another approach to establish parthenocarpy in plants is the increase ofthe content and/or activity of at least one gibberellin in the cells ofthe female reproductive organ. This might be achieved, for example, byoverexpression of genes coding for proteins involved in the biosynthesisof gibberellins in plants. Such genes are described, for example, in Sunet al. (Plant Cell 4 (1992), 119-128) and Bensen et al. (Plant Cell 7(1995), 75-84).

The above described high specificity of activity of the DefH9 promoterin the placenta and/or ovules of plants makes it also a useful tool forthe generation of female sterile plants which are of relevance, forexample, in the generation of hybrid seeds. Thus, in another aspect thepresent invention relates to the use of the promoter region of the DefH9gene of Antirrhinum majus or of a promoter region of a gene homologousto the DefH9 gene of Antirrhinum majus and capable of directing placentaand/or ovule specific expression of a DNA sequence linked to it inplants for the establishment of female sterility. As described in EP-A10 412 006, female sterility can be achieved by expressing specificallyin cells of the female reproductive organs of a plant a so-called“female-sterility DNA”, i.e. a DNA which upon expression significantlydisturbs adversely the proper metabolism, functioning and/or developmentof a cell of a female reproductive organ. Preferably said DNA leads uponexpression in a cell to cell death. Examples for such DNA sequences,which in combination with the DefH9 promoter region might be used toestablish female sterility in plants are: DNases (e.g. endonucleasessuch as restriction endonucleases), RNases (e.g. Bamase or RNase T1),proteases (e.g. trypsin or papain), glucanases, lipases, lipidperoxidases, plant cell wall synthesis inhibitors or bacterial toxinswhich are toxic to plant cells (e.g. botulin, diphteria toxin etc.).Other examples are DNA sequences which encode an antisense RNA moleculeof a transcript of a gene normally expressed in the plant cells. Thisapproach is described, for example, in EP-A 0 223 399. The transcribedantisense RNA inhibits the translation of a corresponding transcriptnaturally occurring in plant cells. DNA sequences which might be used inthis regard comprise preferably those which encode for RNA or proteinswhich are essential for plant cell functioning, e.g. rRNA genes. Anotherexample for a DNA sequence which might be used as a“female-sterility-DNA” is a DNA sequence which encodes a ribozymecapable of highly specifically cleaving a given target sequence. Such atarget sequence is again preferably a transcript of a gene the productof which is essential for plant cell functioning.

As is evident from the above, the specificity of expression and theundetectable constitutive level of expression in vegetative tissue playa crucial role in the generation of transgenic plants unaltered in theirvegetative growth and yet displaying parthenocarpic development orfemale sterility. The DefH9 gene is among the flower-specific genestested so far, the only one displaying an undetectable level ofexpression in vegetative tissues. This requirement is essential whenusing a gene the expression of which influences the auxin content and/oractivity in plants or leads to the killing or disabling of cells. Forexample, an increase of auxin content and activity within the ovaryachieved by ovule-specific expression is bound to mimic the exogenoussubministration of auxin used in horticultural practice. However, even avery low basal expression within vegetative tissue is bound to interferewith plant regeneration and/or plant growth. A modification ofvegetative growth, however, is an undesired trait in horticultural andfruit crops.

In yet a further aspect, the present invention also relates torecombinant DNA molecules comprising a DefH9 promoter and linked theretoat least one DNA sequence which leads when expressed in plants to theincrease of the content and/or activity of at least one auxin in thecells. Such a recombinant DNA molecule comprises in general furthermorea transcriptional termination region at the 3′ end of said DNA sequence.

In a preferred embodiment the promoter used in such a recombinantmolecule comprises the nucleotide sequence as set forth in SEQ. ID No. 1or a fragment thereof which still confers specific expression in theplacenta and ovules of transgenic plants.

The DNA sequence linked to said promoter in such a construct ispreferably one of the DNA sequences mentioned above in connection withthe use of a DefH9 promoter for the generation of plants showingparthenocarpic development.

In a further aspect, the present invention also relates to recombinantDNA molecules comprising a DefH9 promoter and linked thereto at leastone DNA sequence which leads when expressed in plants to the killingand/or disabling of cells of the female reproductive organ of the plantsthereby resulting in female sterility. Such a recombinant DNA moleculecomprises in general furthermore a transcriptional termination region atthe 3′ end of said DNA sequence.

In a preferred embodiment the promoter used in such a recombinantmolecule comprises the nucleotide sequence as set forth in SEQ. ID No. 1or a fragment thereof which still confers specific expression in theplacenta and ovules of transgenic plants. The DNA sequence linked tosaid promoter in such a construct is preferably one of the DNA sequencesmentioned above in connection with the use of a DefH9 promoter for thegeneration of plants showing female sterility.

Furthermore, the present invention also relates to host cellstransformed with a recombinant DNA molecule as described abovecomprising a DefH9 promoter and linked thereto a DNA sequence whichleads when expressed in plants to the increase of the content and/oractivity of at least one auxin in the cells. The host cells can beprokaryotic, for example, bacterial, or eukaryotic, for example fungalor animal cells. In a preferred embodiment, the host cells are plantcells. Thus, the present invention also relates to transgenic plantcells transformed with and genetically engineered with a recombinant DNAmolecule comprising a DefH9 promoter and linked thereto a DNA sequencewhich leads when expressed in plants to the increase of the contentand/or activity of at least one auxin in the cells. Such cells arecharacterized by the feature that they contain stably integrated intotheir genome a recombinant DNA molecule as described above.

In a preferred embodiment of the present invention the plant cells aretransformed with an iaaM gene as well as with a rolB gene, both underthe control of a DefH9 promoter.

The present invention also relates to transgenic plants comprising plantcells according to the invention. Such plants can show parthenocarpicdevelopment due to the increase in auxin content and/or activity withoutalterations of their vegetative growth. This last feature isparticularly relevant and it is due to the absence of detectableexpression in vegetative tissue of the DNA sequence linked to the DefH9promoter. Preferably the plants according to the invention show at leastone of the following features:

a) parthenocarpic development (i.e. fruit development in the absence offertilization);

b) seedless parthenocarpic fruits;

c) fruits with seeds when pollinated;

d) high specific expression, within the ovary, in the placenta, and inthe ovules, and preferably also in tissue derived from the ovules or theplacenta;

e) no expression in other tissues as deduced from the detection limit ofNorthern blot analysis with probes labeled at a specific activity ofapproximately 2×10⁹ cpm/microgram of DNA;

f) an enhancement of fruit setting and/or development in comparison tocorresponding non-transformed plants.

The present invention also relates to parts of the plants according tothe invention which parts comprise the above-described cells. Inparticular, the present invention relates to the fruits of these plantsas well as to any kind of propagation material, for example, seeds,seedlings or cuttings.

Furthermore, the present invention also relates to host cellstransformed with a recombinant DNA molecule comprising a DefH9 promoterand linked thereto a DNA sequence which leads when expressed in plantscomprising such cells to the killing or disabling of cells of the femalereproductive organs of plants so as to render the plant female sterile.The host cells can be prokaryotic, for example, bacterial cells, oreukaryotic, such as fungal or animal cells. In a preferred embodiment,the host cells are plant cells. Thus, the present invention also relatesto transgenic plant cells transformed with and genetically engineeredwith a recombinant DNA molecule comprising a DefH9 promoter and linkedthereto a DNA sequence which leads when expressed in plants comprisingsuch cells to the killing or disabling of cells of the femalereproductive organs of plants so as to render the plant female sterile.Such cells are characterized by the feature that they contain stablyintegrated into their genome a recombinant DNA molecule as describedabove.

The present invention also relates to transgenic plants comprising plantcells as described above. Such plants show female sterility due to theexpression of the DNA sequence linked to the DefH9 promoter in cells ofthe female reproductive organs.

Furthermore, the present invention relates to parts of theabove-described plants which comprise plant cells according to theinvention and, in particular, to propagation material, for example,pollen, cuttings etc.

Plants according to the invention can belong to any desired plantspecies, preferably used are plants of a family selected from the groupconsisting of Solanaceae, Cactaceae, Papilionaceae, Actinidiaceae,Cucurbitaceae, Rubiaceae, Moraceae, Rutaceae, Vitaceae, Ebenaceaea,Crassularaceae, Rosaceae, Drupaceae, Passifloraceae, Caricaceae,Ericaceae, Gramineae, Cruciferae, Cariofillaceae, Amaryllidiaceae,Iridaceae, Leguminosae, Liliaceae, Paeoniaceae, Papaveraceae,Primulaceae, Scrophulariaceae, Violaceae, Malvaceae and Graminaceae orfrom the species Actinidia sinensis. Plants derived from eggplant,tomato, melon or watermelon, cucumber, citrus species, pepper,strawberries, grapes, apple, pear, cherry or olive are especiallypreferred. In principle both dicotyledons and monocotyledons aresuitable starting materials for the preparation of the plants of theinvention.

The above described transgenic plants of the invention can be used as asource of explants (i.e. ovary and/or ovules) for the production ofhaploid or double-haploid gynogenetic plants. Auxins are growth factorsincluded in the media employed to obtain in vitro gynogenetic callus orembryos and, then, plants. The specific expression of appropriatechimeric genes in the ovules and placenta of the transgenic plants, forexample, the DefH9-iaaM chimeric gene by itself, the DefH9-rolB chimericgene by itself or a combination of the iaaM and rolB coding regionsunder the control of DefH9 sequences, ensures an increase of auxinswhich allows either to improve the frequency of gynogenetic plantsproduced in the plant species where this technique is currently used orto widen the number of species where gynogenesis might be used. Anincrease of auxin content and activity within the ovary achieved byovule-specific expression improves also the recovery of gynogeneticplants following pollination with incompetent pollen by reinforcing thestimulation of the haploid cells division in the embryo sac otherwisecaused only by pollen tube growth and fertilization (for a review see:Keller and Korzum, in: Jain, Sopory and Veilleux (Eds); In vitro haploidproduction of higher plants, Vol. 1 (1996), 217-235; Kluwer AcademicPublishers; The Netherlands). The above mentioned mechanism ofovule-specific gene expression of iaaM and/or rolB coding regions willexert a general positive effect on the frequency of viable embryosformed following sexual hybridization through in vitrostigmatic/placentar/ovular/pollination or gametic fusion/fertilizationaimed at hybridization between distant plant species. As reported byBhojwani and Raste, in; Jain, Sopory and Veilleux (Eds); In vitrohaploid production of higher plants, Vol. 1 (1996), 237-262; KluwerAcademic Publishers; The Netherlands). most of the culture mediaemployed contain auxins as growth factor to stimulate zygote division.The transgenic plants of the invention can further be used for breedingplants with parthenocarpic development, or to increase by breeding theexpressivity of the parthenocarpic trait in varieties showing partialparthenocarpic development. The transgenic plants can be also used as asource of material for gynogenesis and/or in vitro fertilization.

The transgenic plant cells and plants can be prepared according toconventional methods known in the art. In particular, the plants can beproduced by any of the following processes:

1. Transformation of plant protoplasts or plant explants withAgrobacterium tumefaciens bacteria or Agrobacterium rhizogenes bacteriawhich contain a plasmid carrying a recombinant DNA molecule according tothe invention or combinations of at least two recombinant DNA moleculesof the invention.

2. In planta transformation by using Agrobacterium strains harbouringthe aforementioned constructs to infect plants.

3. Electroporation of plant protoplasts, tissues or organs.

4. Bombardment of plant tissues or organs by using particles coated withthe aforementioned recombinant constructs.

5. Plant protoplast uptake of the aforementioned recombinant constructsby using chemical methods (e.g. PEG).

6. Microinjection of the aforementioned recombinant plasmids into plantprotoplasts, meristem, microspore, pollen, ovule, tissue or organ.

7. Fertilization of plants with a mixture of pollen and plasmidscarrying any of the aforementioned.

8. Exposure of whole plantlets or seeds to Agrobacteria harbouring anyof the aforementioned constructs.

9. Introgression of whole or parts of chromosomes containing theaforementioned recombinant constructs.

10. Fiber carbide plant transformation by using the aforementionedconstructs.

11. Fertilization of plants with pollen transformed with any of theaforementioned constructs by means of direct or mediated transformation.

In case that it is intended to introduce combinations of theaforementioned constructs of the invention into the plant cells orplants, the combination can be achieved: (i) by co-transformation ofindependent constructs; (ii) by harbouring on the same construct bothchimeric genes (i.e. DefH9-iaaM and DefH9-rolB genes); (iii) by buildinga bicistronic mRNA containing an internal entry site for ribosomesbetween the two open reading frames in order to ensure that both areexpressed under the control of the DefH9 promoter and controllingsequences; or (iv) by sexual and asexual hybridization of twoindependent transgenic plants containing either, for example, theDefH9-iaaM or DefH9-rolB genes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: A schematic representation of the chimeric genes used. DNAsequences from Antirrhinum majus provide promoter and regulatorysequences for transcription of coding regions from: (i) the iaaM gene ofPseudomonas savastanoi; (ii) the rolB gene of Agrobacterium rhizogenes;(iii) ill1, ill2 or ilr1 genes of Arabidopsis thaliana. When indicatedsignals of termination of transcription from the nos gene of A.tumefaciens were included.

FIG. 2: DNA sequence of the promoter and regulatory sequences of theDefH9 gene of Antirrhinum majus used for the construction of theplasmids as described in the examples

FIG. 3: Northern blot analysis showing the expression of DefH9 mRNA inAntirrhinum majus. 2 μg mRNA from various organs were loaded in eachlane indicated by numbers. From left to right: seedling (1), leaf (2),bract (3), sepal (4), petal (5), stamen (6) and carpel (7). Ahybridization signal is detected only in the case of mRNA extracted fromcarpel.

FIG. 4: Comparison of a wild type SR1 tobacco plant (left) and atransgenic DefH9-iaaM parthenocarpic plant (right). The transgenic plantis unmodified in its vegetative growth, i.e. it does not show anymodification of the growth habit and flowering.

FIG. 5: Comparison of a wild type SR1 tobacco plant (left) and atransgenic DefH9-iaaM plant (right) at flowering. The plant transgenicfor the DefH9-iaaM gene does not show any modification of the growthhabit.

FIG. 6: A comparison of capsules obtained by self-pollination in wildtype and DefH9-iaaM tobacco plants. The transgenic plant is able todevelop a normal capsule when pollinated.

FIG. 7: A comparison of capsules developed by pollination and withoutpollination (parthenocarpic) in DefH9-iaaM plants. The transgenic plantis able to develop capsules when emasculated, i.e. parthenocarpically.The capsules are seedless.

FIG. 8: Capsule from a pollinated DefH9-iaaM transgenic plant. Normalseed development is observed.

FIG. 9: Capsule from the same plant developed from an emasculatedflower. Development of the seeds is not taking place.

FIG. 10: FIG. 10 shows transgenic eggplants transformed with theDefH9-iaaM gene bearing fruits developed by self-pollination ({circlearound (X)}) and without pollination (EM).

FIG. 11: FIG. 11 shows a comparison of fruits obtained byself-pollination and without pollination in an eggplant transformed withthe DefH9-iaaM gene.

FIG. 12: FIG. 12 shows a comparison of fruits in an eggplant transformedwith the DefH9-iaaM gene obtained by self-pollination which containsdeveloping seeds and without pollination which does not contain seeds(parthenocarpic).

DETAILED DESCRIPTION OF THE INVENTION

In the examples the following materials and methods have been used:

1. Bacterial Strains and Cultures

E. coli. cells DH5α were used for propagating the recombinant plasmids.All recombinant plasmids were introduced either by electroporation or byconjugation into Agrobacterium tumefaciens strain GV3101 using standardtechniques (Koncz and Schell, Mol. Gen. Genet. 204 (1986), 383-396).

2. Construction of Recombinant Plasmids and Constructs

The used recombinant plasmids and constructs were constructed accordingto established methods (Maniatis et al., Molecular Cloning, a LaboratoryManual, Cold Spring Harbour Laboratory, 1982).

3. Plant Tissue Cultures and Transformation

3.1 Transformation of Tobacco

Agrobacterium tumefaciens mediated transformation of tobacco plants cvPetit Havana SR1 were obtained using the leaf-disk method. Leaf-diskswere prepared from in vitro-grown plants and pre-cultured for two daysin MS (Murashige and Skoog, Physiologia Plantarum 15 (1962), 473-497)basal medium supplemented with 0.5 mg/l thidiazuron and 0.1 mg/l IAA,were dipped for 5 min. in an overnight agrobacteria culture resuspendedat 0.1 OD₆₀₀ in liquid MS basal medium containing 200 μM acetosyringoneand placed back in the same petri dish. After two days the leaf-diskswere cultured in the same medium containing 100 mg/l kanamycin and 500mg/l cefotaxime and subcultured every three weeks. Developed shoots wererooted in MS hormone-free medium containing 50 mg/l kanamycin. Afteracclimatization the plants were grown under greenhouse condition.

3.2 Transformation of Eggplant

For transformation the female parent of the F1 eggplant hybrid ‘Rimina’released by Istituto Sperimentale per l'Orticoltura was employed. Theprocedure for eggplant transformation was essentially as described inRotino and Gleddie (Plant Cell Rep. 3 (1990), 26-29) and Rotino et al.(Proc. VIIIth Meeting on genetics and breeding of Capsicum and eggplant;Rome, Italy, Sep. 7-10, 1992, Capsicum Newsletter, special issue,295-300) with modifications. Leaf, cotyledon and hypocotyl explants werepre-cultured for two days in MS macro and micro nutrients (Murashige andSkoog, (1962), loc.cit.), Gamborg vitamins (Gamborg et al., Exp. CellRes. 50 (1968), 151-158), 0.5 gl⁻¹ MES, 20 μM acetosyringonesupplemented with the growth regulators (mgl⁻¹) 0.5 ZEA, 0.3 BAP, 0.2KIN and 0.1 NAA, media were solidified with 2 gl⁻¹ phytagel (Sigma), pH5.8. For explant infection, an overnight Agrobacterium tumefaciensliquid culture was centrifugated and the pellet resuspended at 0.1 OD₆₀₀density in MS basal medium, 2% glucose, 200 μM acetosyringone pH 5.5.The cut edges of the hypocotyls were cut again and all the explants wereinfected by dipping in bacteria suspension for 5 min, blotted dry ontosterile filter paper and placed back in the same plates. After 48 h theexplants were transferred to selective medium (described above) withoutacetosyringone and supplemented with 30 mgl⁻¹ kanamycin and 500 mgl⁻¹cefotaxime. Shoot-bud differentiation and shoot elongation was achievedby transferring calli with compact green nodules to the same selectivemedium without NAA. Shoots were rooted and propagated in V3 medium(Chambonnet, “Culture d'anthères in vitro chez trois Solanaceesmaraichères: le piment (Capsicum annum), I'aubergine (Solanummelongena), la tomate (lycopersicon esculentum)” (1985) Thèse de Docteurd'Université, Academie de Montpellier) without antibiotics. Transgenicplantlets were grown in the greenhouse.

4. Detection of Parthenocarpic Fruit Development

4.1 In Tobacco

Flowers produced by two inflorescences of each of ten chosen transformedand one untransformed regenerated plants were daily (for one month)emasculated at a length of 2-3 cm and covered with paper bags. Anotherinflorescence was not emasculated and covered to obtain selfed seeds.

4.2 In Eggplant

Flower buds of transformed and untransformed eggplants were covered withpaper bags. For each plant flowers were either emasculated beforedehiscence of anther (closed flowers) or manually self-pollinated(opened flowers) and covered with a paper bag. The experiment wascarried out at Montanaso Lombardo during the period of November 1996 toJanuary 1997 inside a double plastic greenhouse. This means that theexperiment was carried out under short day condition with low naturallight intensity without artificial illumination and indoor temperatureranging from about 14° C. to about 22° C. These conditions representvery adverse climatic conditions for eggplant fertilization and fruitdevelopment. Inside the greenhouse the average temperature was 16.9° C.which represents very limiting conditions for eggplant fruit setting.More importantly, light duration was only 7 hours per day and itsintensity was only 46.4 W/m².

5. DNA Analysis by PCR

Genomic DNA was obtained from leaf tissue according to Doyle and Doyle(1987). 100 ng DNA were analyzed by PCR by using the 5′ATGATTGAACAAGATGGATTGCACGCAGG 3′ (Seq ID No. 2) and 5′GAAGAACTCGTCAAGAAGGCGATA 3′ (Seq ID No. 3) primers which amplified afragment of 739 bp of the nptII coding region and 5′TCTTGGCTTGTAATGGGGATCC 3′ (Seq ID No. 4) and 5′ GGGTGAATTAAAATGGTCATACAT3′ (Seq ID No. 5) primers which amplified a fragment of 450 bp ofDefH9-iaaM gene. PCR reactions were performed in 25 μl of final volumecontaining 1× buffer (Perkin Elmer), 100 μM dNPTs, 50 pM of each primers1 U Taq polymerase. Forty-five cycles at an annealing temperature of 55°C. were employed.

6. RNA Analysis

Flower buds (0.4-0.6 cm long) were harvested, frozen in liquid nitrogenand mRNA was extracted and purified according to well establishedmethods. The complementary cDNA was synthesized by using iaaM specificprimers (5′ GGGTGAATTAAAATGGTCATACAT 3′; Seq ID No. 5 and 5′TTCTTTGGAACTCGTGTTGAGCTC 3′; Seq ID No. 8) and reverse transcriptase for1 hour at 45° C. The cDNA was used as template for a first PCR reaction,performed at 53° C. for 35 cycles using the same iaaM specific primerand a primer located in the untranscribed but untranslated leadersequence of DefH9 gene at the 5′ end of the intron. An aliquot of thisreaction was used as template for a second PCR assay. This assay wasperformed at a Tm of 55° C. using a “Hot Start” to initiate the assay.The reaction was repeated for 35 cycles. PCR products were analyzed byagarose gel electrophoresis, restriction analysis and DNA Blot withsubsequent hybridization. The reaction products were also sequenced.

7. Northern Blot Analysis

Total RNA was extracted from plant organs and tissues as described byLogemann et al. (Anal. Biochem. 163 (1987), 16-20). Poly A+ RNA wasisolated from total RNA using oligo d(T) Dynabeads (Dynal) following theprotocol of the manufacturer. The amount of RNA was determinedspectrophotometrically. 2 μg mRNA were loaded per lane and seperated ona 1.2% agarose gel containing 7% formaldehyde. The RNA was transferredto Hybond N filters (Amersham) by standard techniques (Maniatis et al.,loc. cit.). Hybridisation with radioactively labeled probes wasovernight at 42° C. in 5×SSPE and 50% formamide. As a probe a 600 bpfragment of the DefH9 cDNA without the conserved MADS box at the 5′endwas used to avoid cross-hybridisation with other MADS box RNAs. Signalswere detected using Kodak X-OMAT AR5 X-ray films.

EXAMPLE 1

Construction of the Plasmid pPCV002-DefH9-iaaM

The recombinant plasmid pPCV002-DefH9-iaaM was obtained by ligating theEcoRI-KpnI fragment of 3480 (14+2250+1212+4 bp) spanning the promoter(2250 bp) and regulatory sequences (1212 bp) of the gene DefH9 fromAntirrhinum majus to the KpnI-SphI fragment of 1775 bp (2+53+1671+49)spanning the coding region of the iaaM gene from Pseudomonas syringaepv. savastanoi. The iaaM gene has been characterized by Yamada et al.(1985) and the sequence used contains 1773 bp (53+1671+49) from the DraIsite located 53 bp before the ATG initiation codon till the SphI site 46bp after the TAA stop codon. The transcription termination sequencesfrom the nopaline synthase gene of Agrobacterium tumefaciens are used atthe 3′ of the coding region. The DNA sequence used is 222 bp long fromthe site SphI to the site HindIII, both provided by linker sequences.Consequently, the plasmid pPCV002-DefH9-iaaM obtained possesses thefollowing structural features:

2250 bp of the DefH9 promoter from Antirrhinum majus (having an EcoRIadapter of 14 bp at the 5′ end);

1212 bp of regulatory sequences present in the transcribed butuntranslated leader sequence of the DefH9 gene from Antirrhinum majus(including the intron of 1045 bases);

6 bp added by Kpn I linker addition;

53 bp of untranslated sequence of the iaaM gene from Pseudomonassyringae pv. savastanoi with the nucleotide sequenceCTGAGGTACCGAAAGAATCG (Seq ID No. 6) at the fusion site (fusion obtainedby adding a KpnI linker)

1671 bp of coding region of the iaaM gene from Pseudomonas syringae pv.savastanoi;

49 bp (46+3 bp of stop codon) of 3′ untranslated trailer sequence of theiaaM gene from Pseudomonas syringae pv. savastanoi; and

222 bp containing transcription termination sequences from the nos geneof Agrobacterium.

EXAMPLE 2

Construction of the Plasmid pPCV002-DefH9-rolB

The recombinant plasmid pPCV002-DefH9-rolB was obtained by ligating theEcoRI-KpnI fragment of 3480 bp spanning the promoter and regulatorysequences of the gene DefH9 from Antirrhinum majus to the KpnI-HindIIIfragment of 1509 bp (39+774+694+2 extra bases added with the Kpn Ilinker) spanning the coding sequence of the rolB gene. Consequently, theplasmid pPCV002-DefH9-rolB obtained possesses the following structuralfeatures:

2250 bp of the DefH9 promoter from Antirrhinum majus (having at the 5′an EcoRI adapter of 14 bp);

1212 bp of regulatory sequences present in the transcribed butuntranslated leader sequence of the DefH9 gene from Antirrhinum majus(including the intron of 1045 bases);

6 bp added by linker addition;

39 bp of untranslated sequence of the rolB gene from Agrobacteriumrhizogenes Ri plasmid A4 with the nucleotide sequence CTGAGGTACCGGGCACTT(Seq ID No. 7) at the fusion site (fusion obtained by adding a KpnIlinker);

774 bp of coding region of the rolB gene from Agrobacterium rhizogenesRi plasmid A4; and

694 bp of 3′ flanking sequences present at the 3′ end of the rolB codingregion (i.e. 691+3 bp of stop codon)

EXAMPLE 3

The Biological Effect of the DefH9-iaaM Gene in Transgenic TobaccoPlants

Tobacco plants transformed with the DefH9-iaaM construct were analyzedfor expression of the iaaM gene. For this purpose RNA was obtained fromflower buds of the transgenic plants and analyzed by RT-PCR. Theexpected product of 168 bp was detected in 10 out of 10 transgenicplants analyzed but not in the negative control (i.e. untransformedtobacco plants). As expected, the fragments contain a Kpn I site asshown by agarose gel electrophoresis and DNA blot analysis. The DNA ofthe RT-PCR product from one transgenic plant was sequenced. The DNAsequence confirmed that the 168 bp fragment corresponds to the splicedmRNA of the DefH9-iaaM gene. Thus, the transgenic plants expressed thegene in immature flower buds and the pre-mRNA is properly spliced.Tobacco plants transgenic for the DefH9-iaaM gene do not show anyalteration of vegetative growth when compared to untransformed tobaccoplants (see FIGS. 4 and 5). Moreover, they show normal capsuledevelopment when pollinated (see FIG. 6). However, when emasculated,transgenic plants expressing the DefH9-iaaM gene in the flower buds showparthenocarpic development, and consequently seedless capsules (see FIG.9). Untransformed tobacco plants (and plants not expressing efficientlythe DefH9-iaaM gene) do not show parthenocarpic development. The traitis transmitted to the progeny in a mendelian fashion.

EXAMPLE 4

The Biological Effect of the DefH9-rolB Gene

None of the 20 transgenic tobacco plants tested which had beentransformed with the DefH9-rolB construct showed parthenocarpicdevelopment by itself. However, these plants can be used to increaseparthenocarpic development in plants transgenic for the DefH9-iaaM geneeither by crossing or by co-introducing the DefH9-rolB and DefH9-iaaMgenes.

EXAMPLE 5

The Biological Effect of DefH9-iaaM Gene in Transgenic Eggplants

Eggplants transgenic for the DefH9-iaaM gene appear phenotypicallynormal in their vegetative growth and floral morphology. They areindistinguishable from untransformed eggplants. Fruit setting and fruitdevelopment was exclusively achieved in the eggplants transgenic forDefH9-iaaM gene (FIG. 10). In fact in the untransformed eggplant bothemasculated and hand-pollinated flowers fell down. In transgeniceggplants expressing the DefH9-iaaM gene the size of fruits obtainedfrom emasculated was slightly smaller compared to the fruits obtainedfrom self-pollinated flowers (FIG. 11). In addition, they showparthenocarpic development when emasculated, and consequently seedlesfruits (FIG. 12). Since the temperatures used in the experiments were atthe minimum value for fruit setting (optimum average temperature is 28to 30° C.) and the light intensity and duration was below the minimumlevel (optimum average light intensity is 500-800 W/m²) to allowpollination, fertilization and fruit setting, these experimental datashow that the introduction of the DefH9-iaaM gene can permit to avoidexogenous hormone application to plants when grown in adverse climaticconditions. Adverse conditions might be either low temperature and/orlow light intensity or high temperature.

8 3480 base pairs nucleic acid single linear DNA (genomic) NO NOAnthirrhinum majus 1 AATTCGGCAC GAGGTCCCTT TCTATTTTTG CACAAAGCGTCTTTTACTCG TATCAAGAAT 60 TTGATTCTAC TTTATTACTC AAATTCGTCA CTTCTCTTACACACACACAC ACACACACAC 120 ACACACACAC ACACATATAT ATATTACACT CCAGCCCTTTGTATCTATCC CATCTTTCTC 180 TTATTAATGA ATGAACCAAT AAATAGACCT CTAACAAATACAGTTTGAGC AGGCTGGTTG 240 TTTAATAAAA TTAATGCTGG TTGTTAATTT AAACTGACATTGTTTTTGCT CAGACACGGC 300 AACCTCTATA GTACAGTTTC TTCTTAGTAT TGAAAATTTAGTTGTGGATT TTTTTTTTAA 360 GAAATACAAT TTACAGCTAT AATGTACAAT GCCAAGAACTACAGTTATTT TTTTAATCAC 420 TGAAATGCTT ATATATATTA AAAAGAATCT AAAGAGGGTCAGCGCAATTA TTAACTTTTT 480 TCTCCTGAAC ATTGACCAAA CTTAATATGT GAAAACAACAAAAATTCATA AGGCAGAGGG 540 ATCATAGTAC AACATTGGAT TTGGTGTGTT ACATATAATTAATTAGACCA GGTCCCCTCA 600 GTTACTATTC ATGTAAAACT TGTACTTATT GAGCAGATATTTCTAAAGCT ATACCCTAAC 660 CAATCAAACT GGACTACGTA CCCTATCCTT TCAAAGGTTTTTTTTTTTTT TTTTTTTTCC 720 TCCCAATTAA ATTCGCGTGC ACAAACAAAA CTATATTAATCAGGTAAGAA AATTGCGACT 780 CATATAGTTT TCCATGTTAA AAAAAGTGAG ATATACCAATTAATTTCACT GCATGCAAAC 840 AATATGCATG CCCAAGTAAG TTATGGAAGT TCTTTTTCCTATATATAGAA ACCAACTTAG 900 CAATCTCTAT TTCATATATA TATATAAACA GTTAATTTATTAGTCTCTGA AAAAATTTAA 960 TGCAAGTCGA TCGGTTTACA AAAAGTATAT ATGGGCAATTAAATTGGAAC AATAAGTGTC 1020 ACGCTAGTTT TGAATCAGCT CATGATCATG ACAGGATACTCCATAAGTTT TCATTAAATC 1080 TTAGCTGATA TATCTAGTTA GGAGCCGTAG ATATATAAGAAGGTAACGAT TAAATTGAAA 1140 CGATAAGTTA CATATTATAA TATGTCATTT GTATGATTACTTGATTAGGG TATTAGATTG 1200 TGCAGCCTAA TGTATTGTAC ATTAATTCCC TCCTTTCTAACACGGTTCAA CTCATGTATA 1260 AAATTTTAGG GGTATTACCG ATAATTCACG TAAAATTATAATTATGATTG TATTCCTAAT 1320 AAAAATAGTC CACTAATGTA CGCAATTGCA ATTGACTCATTGAACATATT GAAAAACTCC 1380 CGGTTCGGCA TGCTGCCTCA AGACACGGTC TCTCTAACGAACCGAATACA CAAATTTATG 1440 TGTGTTTCGT CGCTTTTTGC GTGTACCATA TAATCGGATTGCTTCATAAA GGGAGGTTAA 1500 ATAAACTCTG CTACAATTCA ACCTCAGTAG ATTATTTGATGCGCCAAGCA ACAACGGTTA 1560 TATTATGCAA CGAAGTACGA GCTTATCAAA TTACATTGTTTCGGGCTCAT ATCTCTAATA 1620 GTCCTACTAA ACCCCGTAAT ATATAGCAAA ATAATAGTACACAGATTCAA AAATAAAACC 1680 CCTTAATATG AGGCTACTAT CGACTACCAA AGGTAATACACATCATAATC AATGTTCCAA 1740 AAACATAATT AAAAACAGTT AATTATATTA AGTCCATGTAGTTTTTAAAA TTAAGAGATA 1800 TATTCAAGTC TCAACAAACA CATGCAAGTT ACATATCTAGTGACTTCTGC GTGTAATGCA 1860 CCTAACAACA AACCCTAACC AGCCAAAACT AAAAAATATATATATGTAAC ACAGTAACAG 1920 AATATATTCA CCTCCCAAAA TCCCATTATT TATAAGAATTTTTTTAAAGT TCTTGGTAAT 1980 TAATTCCCGC ATGCAAACTC ACCTAATTTT TTTCTATGCTCACCTGGGAT TTAATAATTA 2040 TAAAAAAGAC ATTAAAACAT TTTACAAAGT CATGCAACAATCCTTTAAAA AAAAAAAAAA 2100 AAAAAGCTGA AGCAATTACT ATATTTGGTG CGAATTCTCCCTGCAGAGCT GATAATAATC 2160 ACACCACGCC TGGTACAAAA ATGGAAATGG TGTCATTTTCTTGGCCAGCT CTTCTATCTC 2220 TCCTTCTTTT GCACTACATA AGATAAAGCT AGGTATATACAAAGAAAGAA AATAAGTATA 2280 TCAAAATAAT TAGTGGTGTG ATTATTTAAT ATTTATTTGATCATTCAAGA AACTAAAAAC 2340 TTTGAAGGGA TTCTTTGGAA CTCGTGTTGA GCTCTCAAAACTCGCCGGAA AATAGAAATA 2400 TTTTCCGAAC AAGACAGGTT TGTGAGTCAT CATGCAGATCATGAAGATTG TCTAATTATA 2460 TATTAAAAAA GGAATAAATA TTTCTTTAAG TATGGATTGGTTAATTAATT TATTTTTTCC 2520 TCTTTATGTT TATGGCACAG TACCAAATGT TTTCTCTTTGTGCTCAAATT TATGTCAGTT 2580 TTTTTTTGTA TGTTCTTGTT TAAGCATGGA TCTATTGCCATAACACATAA AACTTGTTTT 2640 TTGGCTTGAA AGATTTAATC TTTCCTCCTA TTTTTTCATGGGTTTTTTTT TTTTTTTTTT 2700 TTTTATTCAT TGACAAGAAT GTCAAATCTT TAGTATGATTTTTATTTTTA TTTGTATGCA 2760 TGATTTCAAA AGCTTTTAAT TTGCTATCTT CTAGCGCCAAAAACTTGTTT CTACCCTAGG 2820 GGACTATGGA ACTGAGGGGA ATCTTTGGAA ACTTCTGATTTCATTTTGGG CCTTGTTTGT 2880 TTTTCTGATT TCTTGTTTTT GGAGGGGACT TTTATAAAATATGAGCTGTG TAAAGTCGAT 2940 GAAGGAGGTT TTGACTCTGA TCCCTCTTTC AAATTTTGGTTGAGTTAAGC TTTTGAAGTC 3000 ATTAAAAAGA GCTATATATA TCACTGCCAA GAACTTTGCCAAATAGTTTC AAGATATAAT 3060 TTTTTTTAGT TCAAAGAACA TAGTTTTTTG ATCTTGGCTTGTAATGGGGA TCCTGCTTTT 3120 TTTTTTTTTT TTTCAGTTCA AATTAATTTC TCATCTTGCTATTCTTGAGG GGCTAATTAC 3180 AGGATTCTTC AGAAAAAATC ATGTATAAGA TTTTCATTATCTTTTTGTAC ACTATGTATA 3240 GATTTTCAGC TGATTGTTTA TCAAAGCATC CTCTTCAAAAAGTCTTTCTA TTTTCAAATT 3300 AAAACTATGT CTTCTCTGTG TGTGTTGAAT CAAAAGACTTCCTTTTCTTT TTTTTTGCTA 3360 CAAAGAAAGA AAATCCAGTG TTTGCTTTAG ATCTATGATACATTGTTCTC TATGATCAAG 3420 ATTAATAAAT CTTATAGTGA GCTTTTTGTT TATTATGATTAGGTTATTTT TCTGAGGTAC 3480 29 base pairs nucleic acid single linearother nucleic acid /desc = “oligonucleotide” YES NO 2 ATGATTGAACAAGATGGATT GCACGCAGG 29 24 base pairs nucleic acid single linear othernucleic acid /desc = “oligonucleotide” YES NO 3 GAAGAACTCG TCAAGAAGGCGATA 24 22 base pairs nucleic acid single linear other nucleic acid/desc = “oligonucleotide” YES NO 4 TCTTGGCTTG TAATGGGGAT CC 22 24 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” YES NO 5 GGGTGAATTA AAATGGTCAT ACAT 24 20 base pairsnucleic acid single linear other nucleic acid /desc = “oligonucleotide”YES NO 6 CTGAGGTACC GAAAGAATCG 20 18 base pairs nucleic acid singlelinear other nucleic acid /desc = “oligonucleotide” YES NO 7 CTGAGGTACCGGGCACTT 18 24 base pairs nucleic acid single linear other nucleic acid/desc = “oligonucleotide” YES NO 8 TTCTTTGGAA CTCGTGTTGA GCTC 24

We claim:
 1. A method for establishing parthenocarpy or enhancing fruitsetting and development comprising the steps of: a) transforming a plantcell or tissue with a nucleic acid molecule comprising: i) a promotercapable of conferring placenta- and/or ovule-specific expression to aDNA sequence operably linked to it, wherein said promoter comprises anucleic acid sequence that has at least 85% sequence identity tonucleotides 1-2264 of SEQ ID NO: 1; and ii) a DNA sequence operablylinked to the promoter, wherein the DNA sequence encodes a proteininvolved in the biosynthesis of an auxin when expressed in plant cells;and b) generating a plant comprising the transformed plant cell ortissue.
 2. The method of claim 1, wherein the nucleic acid sequence hasat least 90% sequence identity to nucleotides 1 to 2264 of SEQ ID NO: 1.3. The method of claim 2, wherein the nucleic acid sequence has at least95% sequence identity to nucleotides 1 to 2264 of SEQ ID NO:
 1. 4. Themethod of claim 3, wherein the nucleic acid sequence is nucleotides 1 to2264 of SEQ ID NO:
 1. 5. The method according to claim 1 or 4, whereinsaid DNA sequence encodes a protein normally not expressed in plantcells.
 6. The method according to claim 5, wherein said DNA sequenceencodes a bacterial protein.
 7. The method according to claim 6, whereinsaid DNA sequence is the iaaM gene of Pseudomonas syringae or a genecoding for a functionally equivalent protein from another organism.
 8. Arecombinant nucleic acid molecule comprising: a) a promoter capable ofconferring placenta and/or ovule-specific expression to a DNA sequenceoperably linked to it, the promoter comprising a nucleic acid sequencehaving at least 85% sequence identity to nucleotides 1-2264 of SEQ IDNO: 1; and b) a DNA sequence operably linked to the promoter, whereinthe DNA sequence encodes a protein involved in the biosynthesis of anauxin when expressed in a plant cell.
 9. The recombinant nucleic acidmolecule of claim 8, wherein the nucleic acid sequence has at least 90%sequence identity to nucleotides 1 to 2264 of SEQ ID NO:
 1. 10. Therecombinant nucleic acid molecule of claim 9, wherein the nucleic acidsequence has at least 95% sequence identity to nucleotides 1 to 2264 ofSEQ ID NO:
 1. 11. The recombinant nucleic acid molecule of claim 10,wherein the nucleic acid sequence is nucleotides 1 to 2264 of SEQ IDNO:
 1. 12. The recombinant nucleic acid molecule according to claim 8 or11, wherein the DNA sequence encodes a protein which is normally notexpressed in plant cells.
 13. The recombinant nucleic acid moleculeaccording to claim 12, wherein the DNA sequence encodes a bacterialprotein.
 14. The recombinant nucleic acid molecule according to claim13, wherein the DNA sequence is the iaaM gene of Pseudomonas syringae ora gene coding for a functionally equivalent protein from anotherorganism.
 15. A host cell transformed with the recombinant nucleic acidmolecule according to claim 8 or
 11. 16. A plant cell comprising therecombinant nucleic acid molecule according to claim 8 or
 11. 17. Aplant comprising the plant cell according to claim
 16. 18. A part of aplant comprising the plant cell according to claim
 16. 19. The part of aplant according to claim 18, which is a fruit, a seed, or otherpropagation material.
 20. The method of claim 1, wherein the nucleicacid sequence has at least 85% sequence identity to SEQ ID NO:
 1. 21.The method of claim 20, wherein the nucleic acid sequence has at least90% sequence identity to SEQ ID NO:
 1. 22. The method of claim 21,wherein the nucleic acid sequence has at least 95% sequence identity toSEQ ID NO:
 1. 23. The method of claim 22, wherein the nucleic acidsequence is SEQ ID NO:
 1. 24. The recombinant nucleic acid molecule ofclaim 8, wherein the nucleic acid sequence has at least 85% sequenceidentity to SEQ ID NO:1.
 25. The recombinant nucleic acid molecule ofclaim 24, wherein the nucleic acid sequence has at least 90% sequenceidentity to SEQ ID NO:
 1. 26. The recombinant nucleic acid molecule ofclaim 25, wherein the nucleic acid sequence has at least 95% sequenceidentity to SEQ ID NO:
 1. 27. The recombinant nucleic acid molecule ofclaim 26, wherein the nucleic acid sequence is SEQ ID NO:
 1. 28. Avector comprising the recombinant nucleic acid molecule according to anyone of claim 8, 11 or
 27. 29. A host cell comprising the vectoraccording to claim
 28. 30. A plant cell comprising the vector accordingto claim
 28. 31. A plant comprising the plant cell according to claim30.
 32. A part of a plant comprising the plant cell according to claim30.
 33. The part of a plant according to claim 32, which is a fruit, aseed, or other propagation material.